This proposal outlines studies directed towards the cloning, expression, and characterization of novel neurotransmitter transporters with a principal focus on the high-affinity choline transporter. Neurotransmitter transport processes have already been implicated in a number of neurological diseases, including amyotrophic lateral sclerosis, Parkinson's disease, and drug addiction. Knowledge of transporter structure and regulation is needed for further assessment of the molecular basis for these and other neurological diseases, as well as for providing a foundation for rational therapeutic design. Neurotransmitter transporter cDNAs will be identified through the use of a homologous cloning strategy employing polymerase chain reaction (PCR) technology. Regional expression patterns of transporter homologues will be assessed by in situ hybridization in rat brain to allow a preliminary prediction of transporter substrate phenotype. Novel transporter clones will undergo further characterization after functional expression in mammalian cell lines. Preliminary studies demonstrate the feasibility and power of this neuroanatomically-based approach for the identification and characterization of related neurotransmitter transporters. We will focus these studies on tissues and cell lines enriched for the choline transporter in order to narrow the search for a functional choline transporter cDNA. Polyclonal antibodies will be raised by immunization of rabbits with synthetic oligopeptides and fusion proteins corresponding to poorly- conserved regions of the transporters identified and used to compare with the in situ distribution of transporter mRNA in rat brain. After an extensive characterization of antibody specificity, antibodies will be used to localize the transporters in rat brain. Finally, the ability of phosphorylation to affect transporter function will be directly determined.